The inflammatory and counter-inflammatory behavior of the immune system following severe injury governs the clinical trajectory of critically-injured patients. For example, a dominant counter- inflammatory phenotype will suppress normal anti-microbial immunity and predispose the host to opportunistic infections. On the other hand, augmentation of inflammation can promote systemic inflammatory shock if infectious complications occur in injured patients. We have reported that a distinct subset of CD4 T cells called regulatory T cells (Tregs) are activated by injury and act to potently suppress both T cell mediated immune function and injury-induced inflammation. Thus, we suggest that Tregs act as an injury-reactive cell subset with the capacity to control both innate and adaptive immune function. The experiments in this project will test this hypothesis and will uncover the cellular and molecular pathways responsible for the rapid activation of Tregs by injury. The following specific aims are proposed: 1. to determine if Tregs are activated by injury by antigen receptor dependent or independent mechanisms. CD4 T cells are specifically activated by T cell receptor (TCR) binding to antigens presented by cell-surface MHC class II (MHCII) molecules on antigen presenting cells. The experiments in this aim will address the MHCII-dependence on Treg activation using MHCII gene deficient (MHCII-/-) mice, FoxP3-GFP gene knock-in mice, and adoptive transfer approaches. Innate (MyD88 and TRIF) and T cell costimulatory pathways (CD40L, CD28, and CTLA-4) will also be tested as potential MHCII independent pathways for the injury-specific activation of Tregs. 2. To characterize injury-specific activation and signaling by Tregs. The early activation of Tregs by burn injury suggests that Tregs respond rapidly and specifically to injury. In this aim, we propose to identify and study TCR-dependent and TCR-independent signaling pathways that are activated in Tregs by burn injury in mice. The experiments in this aim will use phospho-flow cytometry to measure MHCII-, TLR-, CD28-, CD40L-, or CTLA4-dependent activation of Tregs. 3. To define the specificity and nature of injury-specific recall responses by Tregs. The recent discovery that Tregs display a recall memory-like response to burn injury supports the idea that Tregs may respond specifically to injury. Studies in this aim will further investigate this basic finding and will use this observation to advance our understanding of injury-specific immune responses. The clinical significance of this project is that this information could be used to develop innovative approaches for controlling the immune system complications that occur in critically-injured trauma and surgical patients.